Pasteurellosis vaccines

ABSTRACT

A pasteurella vaccine e.g. for use with cattle or particularly sheep, comprises antigenic material derived from the A1 and A2 serotypes of Pasteurella haemolytica, the A1 serotype antigenic material comprising capsular extract, e.g. especially salicylate extract, and the A2 serotype antigenic material comprising heat-killed whole organisms, and a process for the production of a pasteurellosis vaccine comprises preparing said antigenic material from P. haemolytica organisms and formulating the antigenic material into a vaccine, e.g. especially an aluminium hydroxide gel/oil adjuvant vaccine. Also a method for use in agricultural animal husbandry for the prevention and control of pasteurellosis in sheep, cattle and other animals comprises vaccinating the animals with a vaccine according to the invention.

This invention relates to pasteurellosis vaccines their production anduse for the prevention and control of pasteurellosis in sheep, cattleand other animals.

Pasteurellosis is a common respiratory disease of sheep and cattle whichmay often lead to fatality, particularly in the case of young animals,and thus the prevention and control of this disease is of greatimportance to farmers engaged in the rearing of sheep and cattle. Insheep the disease appears as either a pneumonia or a septicaemiccondition dependent upon the age of the infected animal and the strainof the infecting organism, whereas in cattle the disease is encounteredprimarily as a penumonia in regions with temperate climates. Pasteurellahaemolytica has been identified as the main causative agent of thedisease in sheep, and two of the serotypes of this organism togetherwith strains of an associated organisms, Pasteurella multocida, appearto be responsible for the pneumonic disease of cattle. Two biotypes ofP. haemolytica have been identified, the A biotype generally associatedwith septicaemias in young lambs and pneumonias in older sheep, and theT biotype generally associated with septicaemias in adult sheep, andwithin these two biotypes twelve different serotypes have beenidentified. Only the A biotypes but not the T biotypes of P. haemolyticaappear to be involved in cattle pneumonias.

At present vaccines are commercially available for use in the preventionand control of pasteurellosis in sheep and cattle, the sheep vaccinescomprising formalin killed organisms of a range of serotypes of P.haemolytica. These vaccines, however, do not appear to providesatisfactory protection against pasteurellosis. It has now been foundthat effective pasteurellosis vaccines for sheep and possibly cattle canbe prepared if careful attention is paid to the source and method offormulation of the P. haemolytica antigenic material which isincorporated into the vaccines.

The various serotypes of P. haemolytica as hereinafter referred to e.g.the A1 and A2 serotypes, may be determined by methods previouslydescribed and understood in the art; for instance, as determined by theserotyping methods described by Biberstein et al (Biberstein, E. L.,Gills, M., & Knight H. (1960) Cornell Veterinarian 50, page 283).

According to the present invention a pasteurella vaccine comprisesantigenic material derived from the A1 and A2 serotypes of Pasteurellahaemolytica, in which the A1 serotype antigenic material comprisescapsular extract and the A2 serotype antigenic material comprisesheat-killed whole organisms.

The invention also includes processes for the production ofpasteurellosis vaccines in which antigenic material from the A1 and A2serotypes of P. haemolytica, comprising capsular extract of the A1serotype and heat-killed whole organisms of the A2 serotype, is preparedfrom P. haemolytica organisms and formulated into a vaccine. Furthermorethe invention also includes methods for use in agricultural animalhusbandry for the prevention and control of pasteurellosis in sheep,cattle and other animals which comprises vaccinating the animals with avaccine according to the invention.

Vaccines according to the invention may be used in agriculturalapplications for the prevention and control of pasteurellosis in generalto combat both animal losses and inefficient meat production due to thedisease. In particular vaccines according to the invention are suitablefor vaccination of sheep and cattle. Generally the vaccines may containfurther antigenic material in addition to antigenic material derivedfrom the P. haemolytica A1 and A2 serotypes, and it will be appreciatedthat the nature of such further antigenic material may depend upon thespecies of animal and form of disease for which the vaccine is intended.Thus, in addition to antigenic material derived from the A1 and A2serotypes of P. haemolytica, vaccines for sheep may comprise antigenicmaterial derived from other serotypes of P. haemolytica, and vaccinesfor cattle typically comprise antigenic material derived from strains ofP. multocida. Furthermore, for example, sheep vaccines which areintended specifically to combat only the pneumonic form of the diseasein adult sheep may comprise antigenic material derived from the Aserotypes only of P. haemolytica. Alternatively sheep vaccines intendedfor combatting both the septicaemic and pneumonic forms of the diseasetypically comprise antigenic material derived from a combination of Aand T serotypes.

Preferably sheep vaccines according to the invention comprise antigenicmaterial derived from the A6 serotype of P. haemolytica, and in oneembodiment there is provided a trivalent vaccine comprising A1, A2 andA6 serotype antigenic material for use in combating pneumonias in adultsheep. Alternatively, or preferably in addition, sheep vaccines maycomprise antigenic material derived from the A9 serotype, and especiallyalso the A7 serotype. Furthermore sheep vaccines for combating bothpneumonic and septicaemic pasteurellosis, in addition to additional Aserotype antigenic material e.g. A6, preferably A6+A9, or especiallyA6+A9+A7 serotype antigenic material, comprise T serotype antigenicmaterial, preferably a combination of T3, T4 and T10 serotype antigenicmaterial.

Cattle vaccines typically comprise antigenic material derived fromsuitable strains of P. multocida such as, for instance, types A and D(Carter, G. R. (1955) Am. J. Vet. Res. 18, page 481), preferably acombination of the A and D types of P. multocida.

In addition to the additional antigenic material derived from species ofPasteurellae, the vaccines of the invention may include antigenicmaterial derived from other organisms including other bacteria and alsoviruses. Thus, for example, the vaccine may also include antigenicmaterial derived from viruses associated with Pasteurella organisms inthe aetiology of Pasteurellosis, such as, for instance, antigenicmaterial derived from parainfluenza virus e.g. parainfluenza virus type3 (P13).

The additional antigenic material, besides that derived from the A1 andA2 serotypes, included within the vaccines of the invention, may takeany suitable form, though, preferably is of high immunogenic andantigenic character. Thus the additional Pasteurella antigenic materialmay comprise killed whole organisms of P. haemolytica and/or P.multocida, preferably heat-killed whole organisms. Especially, however,the additional antigenic material comprises an extract of P. haemolyticaand/or P. multocida, e.g. a capsular extract. In this latter respect ithas been found, according to the present invention, that capsularextracts of P. haemolytica generally exhibit advantageous antigenicproperties, particularly desirable for vaccine formulation, as comparedwith killed whole organisms, such as formalin killed whole organisms. Itis thus particularly surprising that heat-killed whole organisms of theA2 serotype of P. haemolytica provide a material having significantlybetter antigenic properties than a capsular extract.

Furthermore, however, in particular embodiments of the present inventionit may be desirable to include A2 capsular extract antigenic material inthe vaccine together with the A2 antigenic material comprising heatkilled whole organisms. It is believed that such combination of capsularextract and heat killed whole organisms of P. haemolytica A2 serotypemay give rise to enhanced antigenic and immunogenic properties ascompared with vaccines containing A2 heat killed whole organism alone,in particular for use in vaccines containing antigenic material derivedfrom several serotypes e.g. at least three, of P. haemolytica and/orother organisms.

The capsular extract antigenic material of the vaccines of the inventiontypically comprises polysaccharide capsule material and may be preparedas an extract from whole cells by any suitable extraction procedure suchas those which are known to workers skilled in the vaccine productionand immunology art e.g., extraction with saline solution. Preferably,however, the capsular extract antigenic material comprises protein andespecially also lipopolysaccharide antigenic material in addition tocapsule polysaccharide antigenic material. It is believed that inclusionof these additional protein and lipopolysaccharide cell components inthe capsular extract gives rise to enhanced antigenic and immunogenicproperties as compared with capsular extracts containing predominantlyonly polysaccharide capsule material. Capsular extracts containing theseadditional components may be prepared from cells by use of suitableextraction treatments, such as use of thiocyanate solutions, Cetavlon orCetrimide. A particularly preferred extraction treatment for preparationof protein and lipopolysaccharide containing capsular extracts for usein the vaccines of the invention is treatment with salicylate e.g.aqueous sodium salicylate.

In the preparation of vaccines of the invention the appropriateorganisms are grown or otherwise obtained, the corresponding antigenicmaterial is obtained from the organisms and is formulated into thevaccine. Capsular extract antigenic material may be obtained from theorganisms by any suitable technique. For example, whole organisms aretreated with salicylate e.g. sodium salicylate at concentrations in therange from about 0.1 M up to about 10 M, especially about 1 M,preferably with agitation, for a period of from about 1 up to about 5hours, especially about 3 hours, cell debris is removed e.g. bycentrifugation, and the soluble antigenic product is purified andconcentrated. Antigenic material comprising killed whole organism may beprepared from live organisms by any suitable method including use offormalin treatment. Preferably, however, or in the specific case of theA2 serotype antigenic material, whole organism antigenic material isprepared by heat-treatment of live organisms. For example, the liveorganisms are heat treated at a temperature of at least 50° C. for aperiod of at least 10 minutes, preferably at least 16 minutes e.g. atabout 60° C. for about 16 minutes.

The antigenic material, as prepared above or otherwise, is incorporatedinto a vaccine formulation which usually also comprises other componentsincluding adjuvants and other materials, such as preservatives. Anysuitable adjuvant may be used including complete Freund's adjuvant (CFA)or incomplete Freund's adjuvant (IFA). In a preferred embodiment,however, an aluminium hydroxide gel-oil adjuvant has been found to beparticularly suitable for use with the vaccines of the invention,advantageously avoiding some of the undesirable side effects e.g.excessive local inflammation, attendant upon inoculation with vaccinescomprising Freund's adjuvants. For example, an especially preferredadjuvant is an Alhydrogel-oil adjuvant, the antigenic material beingadsorbed on to Alhydrogel and the resultant suspension emulsified with asuitable oil, such as Bayol F preferably containing 10% Aracel.

The concentration of antigenic material in the vaccine may be varied asdesired, for instance, in accordance with the dose rate of the vaccine,and in this respect the normal dose used is generally about 1-2 ml.Generally each dose of vaccine comprises about 0.1 to about 20 mg ofantigenic material, especially from about 0.5 mg up to about 10 mg e.g.about 5 mg, of antigenic material of each serotype included within thevaccine.

For prevention and control of pasteurellosis, e.g. for use inagricultural animal husbandry, the vaccines of the invention areadministered to the animals, e.g. sheep or cattle, usually in the formof a subcutaneous injection. The animals may be vaccinated soon afterbirth to provide the animals with protection against pasteurellosis atan early stage in their lives, though it may be desirable to allow aminimum period of time to elapse after birth and before vaccination toallow the immunological system of the animal to develop more fully theability to respond to the vaccine. For example, in the case of lambs itmay be desirable to allow a minimum period of four weeks to elapsebetween birth and vaccination. Also, vaccination may be carried out atparticular periods of the year to provide protection against customaryseasonal outbreaks of pasteurellosis. For example sheep flocks may bevaccinated in late spring or thereabouts with vaccines according to theinvention comprising P. haemolytica A serotype antigenic material toprovide protection against the outbreaks of pneumonic pasteurellosiswhich customarily occur in sheep flocks during the summer.

The present invention for the first time provides an effective vaccinewhich gives effective protection against pneumonic pasteurellosis insheep. Advantageously vaccines according to the invention may also beused to provide protection against pasteurellosis in general includingseptacaemic pasteurellosis in sheep and pneumonic pasteurellosis ofcattle.

The invention is further described by way of illustration only in thefollowing examples, which relate to the preparation of pasteurellosisvaccines and to the testing of these vaccines in sheep.

EXAMPLE 1

3 monovalent vaccines are prepared from the A2 serotype of Pasteurellahaemolytica and are tested in sheep against challenge by an aerosol ofthe homologous serotype. The three vaccines comprise: 1. heat-killedwhole organism (HKO) in an aluminium hydroxide gel (Alhydrogel)/oiladjuvant (AO), 2. capsule extract in complete Freund's adjuvant (CFA)and 3. the same extract in aluminium hydroxide gel (Alhydrogel)-oiladjuvant (AO). The preparation of the three vaccines is as follows:

Vaccine Preparation HKO in AO

Organisms of P. haemolytica serotype A2, strain FA2 are grown, checkedfor purity and inoculated into Oxoid No. 2. broth which is cultured at37° C. for 18 hours with agitation. The resultant growth is inoculatedinto 1.5 liters of Oxoid No. 2 broth which is then incubated for 6 hoursunder the same conditions. Growth is found to be in the region of 10⁹organisms per ml, determined by viable counts and by means of an EELnephelometer. The bacteria are harvested by centrifugation at 4° C. for20 minutes at 12,000 g. The bacteria are then killed by heat treatmentat 60° C. for 16 minutes.

The optimal concentration of heat killed organism for adsorption on tothe aluminium hydroxide gel (Alhydrogel, Miles Laboratories, Slough,England) is determined by titration according to the manufacturer'sinstructions (Miles Laboratories, Slough, England). The antigens arethen adsorbed on to the aluminium hydroxide gel and the resultantsuspension is emulsified in an equal volume of Bayol F (Esso Petroleum,N.J., USA) containing 10% Aracel A (Sigma Chemical Co., St. Louis, Mo.,U.S.A.).

Capsule extract in AO

Serotype A2, strain FA2, organisms are grown and harvested as in thepreparation of the HKO in AO vaccine described above. After harvesting,however, the bacteria deposit is suspended in 1.0 M sodium salicylate indistilled water to 1/10 of the original volume. The suspension is thenshaken at 37° C. for 3 hours and centrifuged at 2800 g at 4° C. for 40minutes. The supernatant, in dialysis tubing, is then dialysed againstthree changes of phosphate buffered saline (pH 7.2) at 4° C. over aperiod of 48 hours and then concentrated to one third volume bypervaporation at 37° C.

The antigenic activity of the concentrate may be determined by titrationin an indirect haemagglutination test against a standard rabbitantiserum to the homologous serotype of P. haemolytica. The capsuleextract may be freeze dried and stored at -20° C.

As for the HKO vaccine the optimal concentration of capsular antigen isdetermined by titration, the capsular extract adsorbed on to Alhydrogeland the resultant suspension emulsified with an equal volume of Bayolcontaining Aracel to give an A2 capsule extract in AO vaccine.

Capsule extract in CFA

Capsule extract of type A2, strain FA2, organisms of P. haemolytica isprepared as described above. The A2 capsule extract, at the sameconcentration as in the capsule extract in AO vaccine, is thenincorporated by homogenisation in distilled water into CFA (one part ofcapsule extract to one part of adjuvant).

Vaccination and Challenge

Groups of seven 2-3 week old, specific pathogen free (SPF), hysterectomyderived, colostrum deprived lambs were vaccinated by subcutaneousinjection with 2 ml. aliquots of HKO in AO, capsule extract in AO andcapsule extract in CFA vaccine respectively. Eight lambs were leftuntreated to act as controls. The lambs were bled before vaccination, atapproximately 3 week intervals and at necropsy. Eight weeks aftervaccination the lambs were infected intranasally and intratracheallywith log₁₀ 7.2 TCID₅₀ of P13 parainfluenza virus and one week laterexposed for 15 minutes to an aerosol containing 3.1×10⁵ P. haemolyticatype A2, strain FA2, organism per liter. It is estimated that each lambreceived a dose of approximately 1.0×10⁷ organisms.

The lambs were observed for 5 days after exposure to the P. haemolyticaaerosol and clinical scores of the degree of illness suffered wererecorded. 6 of the 29 lambs, four in the capsule extract in AO group andtwo unvaccinated, died or were killed because of severe illness within 5days of challenge. The remaining lambs were killed in random orderbetween 7 and 10 days after challenge, and the lungs of all lambs wereexamined for lesions and the scores recorded.

The basis of the clinical scores and lesions scores are as follows:Dullness, pyrexia (>42.8° C.) or abnormal respiration were assigned onepoint each, and death four points, giving a score for each lamb varyingbetween 0 and 4 on each day. The surface areas of the lesions on thedorsal and ventral aspects of the lung diagrams were measured with aplanimeter and expressed as a percentage: 0=no lesions, 5=up to 10%,10=11 to 25%, 20=>25% of lung surface affected. At the end of theexperiment the daily clinical scores for each animal were addedtogether. Statistical differences between the clinical scores, lunglesion scores and total score (clinical+lesion score) of the groups weredetermined by means of the Mann-Whitney test (Snedecar & Cochrane (1967)Statistical Methods 6th Edn. (page 130)).

The results obtained are given below in Table 1 together with the resultof statistical analysis by the Mann-Whitney ranking test.

As can be seen, there was no statistically discernible differencebetween the clinical scores of any of the vaccinated groups and theunvaccinated controls. However there were significantly lower pneumoniclesion scores in the groups vaccinated with HKO in AO and capsuleextract in CFA. The total scores in the HKO in AO vaccinates weresignificantly different from those of capsule extract vaccinated lambs.

Sera from the lambs was tested for antibodies to P. haemolytica serotypeA2 by the indirect haemoglutination test (IHA) at 3, 6 and 9 weeks aftervaccination and at necropsy. No lambs had detectable antibodies.However, using the Immuno Fluorescent antibody test (FAT), 21 of the 22vaccinated lambs were found to have detectable antibody 6 weeks aftervaccination, and all vaccinates tested at necropsy had titres of between1/256 to 1/2048.

The results obtained overall indicate the superior immunogenicproperties of heat killed whole organisms of A2 serotype as comparedwith capsule extract, and also indicate the existence of a desirablecombination effect of these heat killed organisms with the specificAlhydrogel-oil adjuvant.

                  TABLE 1                                                         ______________________________________                                        Clinical, Lung Lesion and Total Scores in Lambs Inoculated with               P. haemolytica Vaccines and challenged with Homologous Strain                                             Mean   Mean  Mean                                          No. of   No. died or                                                                             Clinical                                                                             Lesion                                                                              Total                                         in lambs killed in Score  Score Score                                Vaccine  group    extremis  (range)                                                                              (range)                                                                             (range)                              ______________________________________                                        HKO                                                                           in                                                                            AO       7        --        5.85   5.0** 10.85**                                                          (0 to 12)                                                                            (0 to (0 to                                                                   10)   22)                                  Capsule                                                                       Extract                                                                       in                                                                            CFA      7        --        5.42   12.1* 17.5                                                             (4-7)  (5-20)                                                                               (9-27)                              Capsule                                                                       Extract                                                                       in                                                                            AO       8        4         12.5   15.6  28.1                                                             (2-20) (5-20)                                                                              (18-39)                              Unvaccinated                                                                           7        2         10.7   18.5  29.2                                                             (4-18) (10-20)                                                                             (16-38)                              ______________________________________                                         *Significant difference (P ≦ 0.05) compared with unvaccinated          control group                                                                 **Significant difference (P ≦ 0.01) compared with unvaccinated         control group                                                            

EXAMPLE 2

A combined heat-killed whole organism (HKO) and capsule extract (CE)monovalent vaccine in an aluminium hydroxide gel/oil adjuvant (AO) isprepared from the A2 serotype of P. haemolytica and is tested in sheep.HKO and CE antigenic material is prepared and formulated into an AOadjuvant vaccine substantially as described in Example 1 to give avaccine containing 2.2 mg of HKO and 5.6 mg of CE antigenic material per2 ml dose. A group of ten lambs are vaccinated with 2 ml doses of thevaccine, and this group together with a control group of ten lambs aresubsequently challenged with an aerosol of A2 organisms and aremonitored for clinical signs of disease and subsequently by post mortemlung examination substantially as previously described. The resultsobtained are given below in Table 2 indicating effective protectionafforded by the combination A2 vaccine.

                  TABLE 2                                                         ______________________________________                                        Clinical, Lung Lesion and Total Scores in lambs inoculated with               P. haemolytica combination HKO and CE vaccine, and in                         unvaccinated lambs after challenge with the homologous strain.                         Clin-             Lung Counts/g                                      Group  No.     ical    Lesion                                                                              Total Lesion Normal                              ______________________________________                                        Vacci- 1       0       5     5     0      0                                   nates                                                                                2       3       5     8     0      0                                          3       0       5     5     0      0                                          4       3       5     8     0      0                                          5       6       5     11    0      0                                          6       11      20    31    1.5 × 10.sup.7                                                                 1.2 × 10.sup.6                       7       6       0     6     --     5 × 10.sup.2                         8       3       5     8     0      0                                          9       0       0     0     --     0                                          10      1       0     1     --     0                                          Mean                                                                          3.3      5*     8.3                                                    Controls                                                                             1       7       10    17    1.6 × 10.sup.5                                                                 2.1 × 10.sup.3                       2       16D     10    26    2.6 × 10.sup.8                                                                 4.3 × 10.sup.7                       3       7       20    27    4.8 × 10.sup.5                                                                 1.5 ×  10.sup.5                      4       0       0     0     --     0                                          5       16D     20    36    5.8 × 10.sup.8                                                                 5.3 × 10.sup.8                       6       17D     20    37    1.8 × 10.sup.8                                                                 1 × 10.sup.8                         7       2       5     7     0      0                                          8       1       5     7     0      0                                          9       1       5     6     1  × 10.sup.3                                                                  0                                          10      17D     20    27    9.3 × 10.sup.7                                                                 1.8 × 10.sup.8                               Mean                                                                          8.4     11.5  19                                               ______________________________________                                         ##STR1##                                                                      D = died                                                                      -- = not done                                                                 *Mann-Whitney P = 0.05                                                   

EXAMPLE 3

Similarly as for previous Examples a capsule extract (CE) monovalentvaccine in an aluminium hydroxide gel/oil (AO) adjuvant is prepared fromthe A9 serotype of P. haemolytica and tested in sheep. The A9 vaccine isprepared and tested against homologous challenge substantially asdescribed in previous Examples, the vaccine containing 5.6 mg of CEantigenic material per 2 ml dose. The results obtained are given belowin Table 3 indicating a high level of protection against homologouschallenge as a result of the use of the A9 monovalent vaccine.

                  TABLE 3                                                         ______________________________________                                        Mean Scores in lambs inoculated with A9 monovalent vaccine                    after homologous challenge compared with Mean Scores in                       unvaccinated lambs.                                                                   Mean Clinical Mean Lesion                                                                              Mean Total                                   Group   Score         Score      Score                                        ______________________________________                                        Vaccinates                                                                            .3*           1.0        1.3*                                         Controls                                                                              2.2           4.4        6.7                                          ______________________________________                                         *Significant at the 5% level; the mean lesion score of vaccinates was         almost significant at the 5% level.                                           ##STR2##                                                                 

EXAMPLE 4 Combined P. haemolytica A1 and A2 serotype vaccine

Organisms of P. haemolytica serotype A2, strain FA2 and serotype A1,strain FA1, were grown and harvested as described previously forserotype A2 in Example 1. Capsule extract was prepared from the A1organisms and A2 organisms were heat-killed, also as describedpreviously. Freeze dried capsule extract of serotype A1 wasreconstituted and incorporated by homogenisation in distilled water intoCFA or IFA (Difco Laboratories, West Molesey, Surrey, England) (1 partof capsule extract to 1 part of adjuvant). Killed whole organisms ofserotype A2 were freeze dried and homogenised in CFA Difco or IFA at therate of 10 mg/ml of vaccine.

Twenty-one SPF lambs between 9 and 25 days old were randomly allocatedinto groups of 10 and 11 lambs respectively. The group of 10 were eachvaccinated subcutaneously with 1 ml of CFA vaccine containing 19 mg/mlof P. haemolytica type A1, capsule extract and 10 mg/ml of type A2 HKO.One month later the same lambs were re-vaccinated with the same antigensin IFA. Four weeks after the second vaccinations both groups of lambswere infected with P13 virus (10⁷.3 TCID₅₀) and one week later with anaerosol of P. haemolytica type A1 and type A2 in equal proportions. Asin Example 1, the lambs were exposed in randomly allocated groups offour for 15 minutes. The atmosphere contained 2.7×10⁵ organisms/liter,with both serotypes present in equal proportions. The lambs wereexamined for signs of clinical illness for a period of 5 days afterinfection with P. haemolytica. Lambs which survived were killed between7 and 10 days after exposure to the aerosol and the lungs of all lambswere examined for lesions. Clinical and lung lesion scores weredetermined as in Example 1 and the results obtained are given below inTable 4.

                                      TABLE 4                                     __________________________________________________________________________    Clinical, Lesion and Total Scores in SPF lambs inoculated with P.             haemolytica                                                                   Vaccine and in unvaccinated lambs after challenge                                        Challenge                                                                            No. of lambs                                                                         Mean Clinical                                                                         Mean Lesion                                                                          Mean Total                                       P. haemolytica                                                                       died or killed                                                                       Score   Score  Score                                 Group      type   in extremis                                                                          (range) (range)                                                                              (range)                               __________________________________________________________________________    A1 capsule Extract                                                            and                                                                           A2, HKO                                                                       (10 lambs) A1, A2 --     5.8**   6.5**  12.3**                                                         (--to 10)                                                                             (--to 20)                                                                            (2 to 29)                             Unvaccinated                                                                  (11 lambs)        6      10.4    20     27.8                                                           (3-14)         (23-34)                               __________________________________________________________________________     **Significant difference (P ≦ 0.01) compared with unvaccinated         control group                                                            

Six unvaccinated lambs died or had to be killed within four days ofchallenge. No vaccinated sheep died. All but one of the vaccinates,however, exhibited some clinical abnormality although their clinicalscores were significantly different (P<0.01) from those of theunvaccinated lambs. All unvaccinated lambs had pneumonic lesions whichinvolved more than one quarter of the lung surface, whereas twovaccinated lambs had no lesions, five had lesions affecting no more than10% of the lung surface and three had more extensive lesions. Both thelung lesion scores and total scores were significantly different(P<0.01) in the vaccinated and unvaccinated lambs.

Vaccination with this combined A1 and A2 serotype vaccine significantlyreduces the severity of pneumonia caused by challenge with a combinationof the same serotypes.

EXAMPLE 5 Combined P.haemolytica A1, A2 and A6 serotype Vaccine

Organisms of P. haemolytica serotypes A1, A2 and A6, strains FA1, FA2and FA6 respectively, were grown and harvested as described in previousExamples, and incorporated in an AO adjuvant vaccine. Heat killed wholeorganisms of type A2 were prepared, also as previously described.Capsule extracts of strains FA1 and FA6 were prepared by sodiumsalicylate extraction. The sodium salicylate extract was centrifuged at40,000 g for 30 minutes and the supernatant dialysed against 0.02 Mphosphate and 0.03 M sodium chloride at pH 7.6 for 48 hours, and wasconcentrated to 1/10 of its volume by ultra-filtration through XM 100 ADiaflo Amicon membrane (Amicon Limited, High Wycombe, Buckinghamshire,England). The concentrate was tested for in vitro antigenic activity byan IHA test and the dry matter concentration determined after furtherdialysis against distilled water. The capsule extracts and HKO wereincorporated into AO adjuvant as described previously, to give a vaccinecontaining 0.18 mg/ml of A1 capsule extract, 0.75 mg/ml of A2 HKO and0.21 mg/ml of A6 capsule extract.

Sixty-four, 2 or 4 weeks old, SPF lambs were randomly allocated intoeight groups, four groups to be vaccinated and paired control groups,such that the age range in vaccinates and controls was similar. Lambs infour groups were vaccinated subcutaneously behind the ear with 2 ml., toeach individual, of A0 vaccine containing 0.37 and 0.42 mg/ml of capsuleextract of strains FA1 and FA6 respectively and 1.51 mg/ml of HKO ofstrain FA2. Eight weeks after vaccination all the lambs were infectedintranasally and intratracheally with 10⁷.7 TCID₅₀ of P13 virus. Oneweek later the groups of lambs were challenged by exposure to an aerosolof P. haemolytica. Vaccinated and paired unvaccinated control groupswere exposed in randomly allocated groups of four to aerosols of strainsFA1, FA2, FA6 and FA9 respectively, the strain FA1 aerosol containing3.8×10⁴, FA2 1.2×10⁴, FA6 1×10³ and FA9 3.1×10.sup. 4 organisms/liter.

Surviving lambs were killed 7 days after challenge. Clinicalexaminations and post mortem examination of lung lesions were assessedas in previous Examples, and the results obtained, together with thestatistical analysis of the results as determined by the Mann-Whitneyranking test, are given below in Table 5.

The results obtained indicate that the combined A1, A2 and A6 serotypevaccine provides good protection against challenge with A1 and A6serotype and also some measure of protection against challenge with theA2 serotype. It is believed that the level of protection against A2challenge may be increased by altering the concentration of the A2 HKOantigenic material in the vaccine.

                  TABLE 5                                                         ______________________________________                                        Clinical, Pneumonic Lesion and Total Scores in lambs inoculated               with P. haemolytica vaccine and challenged as indicated                                                    Mean  Mean  Mean                                                              clinical                                                                            lesion                                                                              Total                                Chal-       No. of  No. died score score score                                lenge       lambs   of killed                                                                              (range)                                                                             (range)                                                                             (range)                              ______________________________________                                        Vaccinated                     5.5*  6.6   12.2                                                              (0-16)                                                                              (0-20)                                                                              (0-36)                                     A1                                                                    Control         8       4      12.8  11.8  24.7                                                              (4-18)                                                                              (5-20)                                                                              (14-38)                            Vaccinated      9       1      4.6   6.6   11.3                                                              (1-14)                                                                              (0-10)                                                                              (0-19)                                     A2                                                                    Control         8       0      9.5   11.2  20.7                                                              (0-10)                                                                              (5-20)                                                                              (5-30)                             Vaccinated      8       0      1.3   2.5*  3.8*                                                              (0-6) (0-5) (0-8)                                      A6                                                                    Control         8       1      4.2   9.3   13.6                                                              (0-11)                                                                              (5-20)                                                                              (0-31)                             Vaccinated      9       2      6.3   10.5  16.8                                                              (3-15)                                                                              (5-20)                                                                              (8-35)                                     A9                                                                    Control         7       3      9.5   10    19.5                                                              (0-17)                                                                              (5-20)                                                                              (5-35)                             ______________________________________                                         *Significant difference at 5% level.                                     

The results obtained in the foregoing Examples are summarised in termsof the ratio of the group mean total score of vaccinates to that ofcontrols in Table 6 below, giving an indication of the relativeprotection afforded by the various vaccines investigated. The result of0.90 for the A9 serotype in the trivalent vaccine case, i.e. Example 5above, arises from the fact that A9 represents a heterologous challengeas it was not present in the vaccine.

                  TABLE 6                                                         ______________________________________                                        PROTECTION EXPRESSED AS: -                                                     ##STR3##                                                                                    VACCINES                                                       MONOVALENT     DIVALENT     TRIVALENT                                         ______________________________________                                                       A1 CE,       A1, A6, CE                                                       A2 HKO CFA   A2 HKO/AO                                         A1 CE/CFA 0.4  A1 0.45      A1 0.45                                           A6 CE/CFA 0.37              A2 0.55                                           A2 HKO/AO 0.38              A6 0.28                                           A2 CE/CFA 0.50              A9 0.90                                           A2 CE/AO 1.0                                                                  A2 HKO                                                                        + A2 CE/AO 0.43                                                               A9 CE/AO 0.19                                                                 ______________________________________                                    

We claim:
 1. A pasteurellosis vaccine comprising antigenic materialderived from the A1 and A2 serotypes of Pasteurella haemolytica, whereinthe A1 serotype antigenic material comprises capsular extract and the A2serotype antigenic material comprises heat-killed whole organisms. 2.The vaccine according to claim 1, comprising additional antigenicmaterial selected from the group consisting of the A6 serotype of P.haemolytica, the A9 serotype of P. haemolytica, the A7 serotype of P.haemolytica and mixtures thereof.
 3. The vaccine according to claim 1 or2 further comprising P. haemolytica T serotype antigenic material. 4.The vaccine according to claim 1 further comprising antigenic materialfrom P. multocida.
 5. The vaccine according to claim 2 or 4 wherein theadditional antigenic material comprises capsular extract.
 6. The vaccineaccording to claim 3 wherein the additional antigenic material comprisescapsular extract.
 7. The vaccine according to claim 1 wherein the A2antigenic material comprises A2 capsular extract together with A2heat-killed whole organism.
 8. The vaccine according to claim 1 whereinthe capsular extract antigenic material comprises protein andlipopolysaccharide antigenic material in addition to capsulepolysaccharide antigenic material.
 9. The vaccine according to claim 8in which capsular extract antigenic material comprises salicylateextract antigenic material.
 10. The vaccine according to claim 1 furthercomprising an aluminum hydroxide gel/oil adjuvant.
 11. The vaccineaccording to claim 1, in dosage form, comprising from about 0.1 up toabout 20 mg of antigenic material per dose.
 12. The vaccine according toclaim 11 comprising from about 0.5 up to about 10 mg of antigenicmaterial per dose of each serotype included within the vaccine.
 13. Aprocess for the production of a pasteurellosis vaccine comprising:cultivating organisms of the A1 and A2 serotype of P. haemolytica;preparing antigenic material of the A2 serotype by heat-killing wholeorganisms of the A2 serotype; preparing antigenic material of the A1serotype by forming a capsular extract from organisms of the A1serotype; and admixing said antigenic materials of the A1 and A2serotypes.
 14. The process according to claim 13 wherein capsularextract antigenic material of the A2 serotype of P. haemolytica isincorporated into the vaccine as well as the A2 heat-killed wholeorganism antigenic material.
 15. The process according to claim 13 or 14wherein capsular extract antigenic material is prepared by salicylatetreatment of organisms comprising: contacting the organisms with asalicylate solution having a concentration in the range from about 0.1 Mup to about 10 M, for a period from about 1 up to about 5 hours;separating cell debris from the solution; and then concentrating andpurifying the soluble antigenic material.
 16. The process according toclaim 15 wherein the salicylate solution contacted with the organismshas a concentration of about 1 M and is contacted with the organisms forabout 3 hours.
 17. The process according to claim 13 wherein theheat-killed whole organisms are prepared by heat treatment of liveorganisms at a temperature of at least 50° C. for a period of at least10 minutes.
 18. The process according to claim 13 wherein the antigenicmaterial is absorbed on to aluminum hydroxide gel which is thenemulsified in oil.
 19. A method for use in agricultural animal husbandryfor the prevention and control of pasteurellosis in sheep, cattle andother animals comprising vaccinating the animals with a vaccineaccording to claim 1 or 2 in an amount effective to controlpasteurellosis.
 20. A method according to claim 19 wherein vaccinationis carried out soon after birth to provide the animals with protectionagainst pasteurellosis at an early stage.
 21. The method according toclaim 20 wherein vaccination is carried out at least four weeks afterbirth.
 22. The method according to claim 19 wherein sheep flocks arevaccinated in late spring with a vaccine comprising said P. haemolyticaA serotype antigenic material to provide protection against summeroutbreaks of pneumonic pasteurellosis.